PROJECT SUMMARY The majority of heterosexually transmitted HIV-1 infections are initiated by a single genetically distinct virus selected from a diverse population of viruses, or quasispecies, in the transmitting partner. This genetic bottleneck is an active area of research, as elucidation of shared properties among viruses initiating infection, or transmitted/founder viruses (TFVs), could indicate viral transmission requirements to target in vaccine development. However, the longitudinal impact of TFVs in HIV-1 infection and disease has been largely neglected as a subject of study. Our lab demonstrated that the TFV replicative capacity (RC), as determined by the transmitted gag gene, is significantly associated with HIV-1 proviral burden in central memory CD4+ T cells (TCM) early in infection. High TFV RC is associated with increased proviral burden in TCM cells, a cell subset implicated as a major component of the viral reservoir, or the population of latently infected cells in which integrated HIV-1 DNA can persist for years even during antiretroviral therapy (ART) treatment. Additionally, viral RC correlates with set point viral load, and is predictive of CD4+ T cell decline to 300 cells or fewer for five years following infection, indicating a potential lasting role for the TFV in disease progression and infection. We hypothesize, therefore, that TFV fitness determines HIV-1 DNA burden in CD4+ central memory T cells during ART-nave infection and impacts the reservoir via persistence of the TFV or similar variants in latently infected cells. We will test this hypothesis in three ways: first we will determine the relationship of TFV replication and HIV-1 DNA load in CD4+ T cells during early and chronic infection of ART-nave individuals, sampling cells from approximately 3 months, 1 year, and 2 years following infection, as well as shortly prior to ART initiation. Second, we will amplify proviral genes from CD4+ T cells analyzed for HIV-1 DNA burden and viral genes from plasma collected during ART-nave chronic infection, then determine if the TFV or closely related variants are present. Last, we will amplify and sequence proviruses from cells collected during ART treatment to determine if the TFV or closely related variants persist, then measure the replication of the viruses to determine the relationship of TFV replication and reservoir variant replication. Our unique collaboration with the Zambia- Emory HIV Research Program (ZEHRP), in which partners of HIV-1 serodiscordant couples (one HIV+, one HIV- partner) are counseled to prevent transmission, the negative partner is monitored for seroconversion, and followed longitudinally in the event of infection, will facilitate these studies. Technical challenges of sequencing and cloning TFVs can be met with molecular cloning and next-generation sequencing technologies developed in our lab. This project will contribute to an understanding of the effect of the TFV and its fitness on HIV-1 infection, including the potential for a TFV influence on the replication of proviruses in the reservoir. Identifying such an impact of TFV fitness would signify that the early stages of HIV-1 infection are a target for therapeutic strategies to modulate viral fitness for constraining the reservoir, a major barrier to HIV-1 cure.